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Titration of Unknown amino acids

(Experiment 2)

By: Racha Al-Khoury

Amino Acids
Building blocks of proteins

http://www.accessexcellence.org/RC/VL/GG/aminoAcid.php

- Amino acid polymerization


R1
H3N+

O-

R2

O
C

O-

H2O
R1
H3N+

O
C

R2
N

O
C

O-

Classification of Amino Acids


Phenyl Group

Nonpolar
(hydrophobic)
Thiol ether

Hydroxyl
Thiol

Acidic
Carboxyl

Hydrocarbon
side chain

Cyclic
secondary
Indole Group
Hydrogen

Polar
(Hydrophilic)
Amine bearing
Phenolic

Basic
Amine

Titration
Common laboratory (volumetric analysis) method of quantitative
chemical analysis that is used to determine the unknown
concentration of a known reactant in which the end point is detected
by the indicator.

This Experiments Objective:

Titrant or Standard

To identify an amino acid by


titrating it with base, while measuring
the pH, and determining its pKa values
from the titration curve.
Analyte or Sample

Titration Apparatus

What is pKa???
pKa1

H+

Zwitterion
pKa: acid dissociation constant

pKa2

H+

All anionic
form
All cationic form
has been reacted

Isoelectrical point
It is the pH at which the number of negative charges equal to the
number of positive charges and the Zwitterionic form predominates.
The relationship between pH and pI:
The pH at which the amino acid is electrically neutral is its isoelectric
point.
The pI for a 1carboxylic-1amino group amino acid is equal to the
mean of the two pKa values ([pKa1+pKa2]/2).

For amino acids with three pKa values, pI = mean of the closest pKa
values (the mean of the two pkas that sandwiches the zwetterion
form)

Titration of Glycine

pI = (pK1 + pK2)

Titration of Histidine

+1

pI = (pKa2 + pKaR)

Todays Experiment
Titration of unknown solution:
1- Place 25ml of unknown solution 1 into a 250ml flask, with a stirrer.
2- Immerse the electrode of the pH meter in the solution and stir with a
magnetic stirrer.
3- Using a plastic pipette, add dropwise 0.1M HCL to the amino acid solution
and adjust its pH if necessary to pH ~ 1.0.
4- Fill the burette with 0.1M NaOH up to the 0 mark.
5- Titrate the mixture with increments of 1ml of the NaOH solution while
stirring and keep reading the volume until the pH rises to 11 (Tabulate the
volume of alkali for each pH change)
6- Plot pH against volume of alkali (Determine pKa values, pI value of the
unknown amino acid solution, and determine the amino acid present)

Todays Experiment
Part A Results:

pH reading
1)
2)
3)
4)
5)
6)
7)
8)
9)
10)
11)
12)
13)
14)
15)
16)
17)
18)
19)
20)
21)
22)
23)
24)
25)
26)
27)
28)
29)
30)
31)
32)
33)
34)
35)
36)
37)

39)
40)
41)
42)
43)
44)
45)
46)
47)
48)
49)
50)
51)
52)
53)
54)
55)
56)
57)
58)
59)
60)
61)
62)
63)
64)
65)
66)
67)
68)
69)
70)
71)
72)
73)
74)
75)

Volume (ml) [increments of 0.5ml]


1)
2)
3)
4)
5)
6)
7)
8)
9)
10)
11)
12)
13)
14)
15)
16)
17)
18)
19)
20)
21)
22)
23)
24)
25)
26)
27)
28)
29)
30)
31)
32)
33)
34)
35)
36)
37)

0.0

39)
40)
41)
42)
43)
44)
45)
46)
47)
48)
49)
50)
51)
52)
53)
54)
55)
56)
57)
58)
59)
60)
61)
62)
63)
64)
65)
66)
67)
68)
69)
70)
71)
72)
73)
74)
75)

Table 3-1: pH readings for Unknown 1

Todays Experiment
14

pH reading

13
12
11
10
9
8
7
6
5
4
3
2
1
0.00

Volume of NaOH added (ml)

Todays Experiment
Amino Acid

pKa
-COOH

pKa
NH3+

pKa
Side Chain

pI

Alanine
Arginine
Asparagine
Aspartic Acid
Cysteine
Glutamic Acid
Glutamine
Glycine
Histidine
Isoleucine
Leucine
Lysine
Methionine
Phenylalanine
Proline
Serine
Threonine
Tryptophan
Tyrosine
Valine

2.34
2.17
2.02
2.09
1.71
2.19
2.17
2.34
1.82
2.36
2.36
2.18
2.28
1.83
1.99
2.21
2.09
2.38
2.20
2.32

9.69
9.04
8.84
9.82
10.78
9.67
9.13
9.60
9.17
9.68
9.60
8.95
9.21
9.13
10.60
9.15
9.10
9.39
9.11
9.62

12.48
3.86
8.33
4.25
6.04
10.79
10.07
-

6.15
10.75
5.40
2.95
5.15
3.10
5.65
6.10
7.65
6.05
6.00
9.80
5.70
5.75
6.30
5.70
5.60
5.95
5.70
6.00

Table 3-3: The pKa & pI values of Amino Acids

The End.